β‐Amyrin synthase

Abstract

β-amyrin, a typical pentacyclic triterpene having an oleanane skeleton, is one of the most commonly occuring triterpenes in nature and is biosynthesized from (3S)-2,3-oxidosqualene. The enzyme, β-amyrin synthase, catalyzing the cyclization of oxidosqualene into β-amyrin, generates five rings and eight asymmetric centers in a single transformation. A homology-based PCR method was attempted to obtain the cDNA of this enzyme from the hairy root of Panax ginseng which produces oleanane saponins together with dammarane-type saponins. Two sets of degenerate oligonucleotide primers were designed at the regions which are highly conserved among known oxidosqualene cyclases (OSCs). Nested PCRs using these primers successfully amplified the core fragment which revealed the presence of two OSC clones PNX and PNY. Specific amplification of each clone by 3′-RACE and 5′-RACE was carried out to obtain the whole sequences. The two clones exhibited 60% amino acid identity to each other. A full-length clone of PNY was ligated into the yeast expression vector pYES2 under the GAL1 promoter to give pOSC PNY . β-amyrin production was observed with the mutant yeast lacking lanosterol synthase, transformed by this plasmid. The sequence of pOSC PNY contains an open reading frame of 2289 nucleotides which codes for 763 amino acids with a predicted molecular mass of 88 kDa. Sequence comparison with other OSCs showed a high level of similarity with lanosterol, cycloartenol and lupeol synthases. The other clone, pOSC PNX , was shown to be cycloartenol synthase by similar expression in yeast. The present studies have revealed that distinct OSC exists for triterpene formation in higher plants, and the high level of similarity with cycloartenol synthase indicates close evolutional relationship between sterol and triterpene biosynthesis.

Cloning and Characterization of Oxidosqualene Cyclases from Kalanchoe daigremontiana

“…The yeast mutant GIL77 (gal2 hem3-6 erg7 ura3-167) was used for this purpose because it is deficient in lanosterol synthase and therefore accumulates high concentrations of 2,3-oxidosqualene that can serve as substrate for OSC-catalyzed cyclization (25). However, growth of this strain requires exogenous ergosterol, which must be added to the medium and is consequently detected in triterpenoid analyses together with the products of the transgenic OSCs.…”

Squalene cyclase and oxidosqualene cyclase from a fern

“…Following the conditions reported in previous papers [18,19], nested PCR was carried out with the single-stranded cDNA mixture as a template and the degenerate primers 162S (5…”

“…The 2.3-kb PCR product was digested with SacI and XbaI and ligated into the sites of pYES2 (Invitrogen) to construct the plasmid pYES2-ACX. Following the same method reported previously [20], the mutant yeast strain GIL77 [18] was transformed with the resultant plasmid pYES2-ACX. The culture conditions of the transformed yeasts, induction protocol, preparation of cell extracts, and analysis using TLC were the same as reported previously [18].…”

Cloning and characterization of a β-amyrin synthase gene from the medicinal tree Aralia elata (Araliaceae)

“…In order to isolate an AeAS gene, specific primers were designed from the sequence of P. ginseng PNY1 (Kushiro et al, 1998a). Both A. elata and P. ginseng belong to the Araliaceae family and they are highly homology.…”